CHEM. RES. CHINESE UNIVERSITIES 2011, 27(2), 258—263

Imprinted Human Serum Albumin with Antioxidant Activity SHEN Na1, YAN Fei1,2, GUO Yi1, LÜ Shao-wu1, GONG Ping-sheng1, XU Ya-wei1, YAN Gang-lin1*, MU Ying1,2* and LUO Gui-min1 1. Key Laboratory of Molecular Enzymology and Engineering, Ministry of Education, College of Life Science, Jilin University, Changchun 130012, P. R. China; 2. State Key Laboratory of Industrial Control Technology, Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, Zhejiang University, Hangzhou 310058, P. R. China Abstract In order to create a new mimic of glutathione peroxidase(GPx), bioimprinting was used to generate glutathione(GSH) binding site and chemical modification was used to incorporate catalytic group selenocystine(Sec). Human serum albumin(HSA) and S-substituted dinitrophenyl glutathione(GSH-S-DNP) were chosen as the imprinted matrix and imprinting template, respectively, to generate a GSH-imprinted protein(GSH-HSA) by bioimprinting. Sec was incorporated into the GSH-HSA by chemical modification to give a new GPx mimic(Se-GSH-HSA). Se-GSH-HSA displayed considerably higher GPx activity than non-printed HSA(Se-HSA). The enzymic properties and kinetics of Se-GSH-HSA were studied. Moreover, Se-GSH-HSA was confirmed to have stronger antioxidant ability to protect mitochondria against oxidative damage with ferrous sulfate/ascorbate-induced mitochondria damage model, indicating that Se-GSH-HSA has potential application in medicine. Keywords Antioxidant; Enzyme mimic; Glutathione peroxidase; Molecular imprinting Article ID 1005-9040(2011)-02-258-06

1

Introduction

Glutathione peroxidase(GPx) is a mammalian antioxidant selenoenzyme which protects biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides(ROOH) with glutathione(GSH) as the reducing substrate[1]. Detailed kinetic studies and modeling of enzyme-substrate complexes have led to the suggestion of the reaction mechanism[2]. However, GPx has some shortcomings such as instability, poor availability and high molecular weight, which have limited its therapeutic application[3]. Thus, some GPx mimics have been prepared. One of them is a selenorganic compound, 2-phenyl-1,2-benziososelenazol-3(2H)-one(ebselen, PZ51), which is the best known GPx mimic[4] and has currently been approved in Japan to treat stroke patients despite its limitations, such as poor water solubility and low activity(0.99 U/μmol) as compared with native GPx. Wilson et al.[5] synthesized some diselenides and

Scheme 1

suggested that the diselenoyl bond should be used as a catalytic moiety for GPx imitation. However, these investigators did not consider the ability of an enzyme model to bind substrate, which generally plays a vital role in enzymatic function. Most existing GPx mimics have low GPx activities because they lack a substrate binding site. To make GPx mimics with higher activity and better specificity, we have prepared a GPx mimic with a GSH binding site using molecular imprinting. Molecular imprinting is a method for making selective binding sites in synthetic polymers via a molecular template. Target molecules can be used as templates for imprinting cross-linked polymers. After the removal of template, the remaining polymer is of more specificity(Scheme 1). The selectivity of the polymer depends on various factors such as the size and shape of the cavity and rebinding interactions. Covalent interactions[6], noncovalent interactions[7], electrostatic interactions[8], and metal ion coordination[9] can be exploited to organize the functional monomers around the template. The

Schematic of molecular imprinting process I. Polymerization; II. template removal/rebinding.

——————————— *Corresponding author. E-mail: [email protected]; [email protected] Received October 8, 2010; accepted November 11, 2010. Supported by the National Basic Research Program of China(No. 2007CB714503), the National Natural Science Foundation of China(No.30970608), the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China (No.2009045), the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China (No.20100948), the Innovation Method Fund of China(No.2008IM040800) and the Open Project of State Key Laboratory of Supramolecular Structure and Materials of China(No.SKLSSM200915).

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SHEN Na et al.

bioimprinting approach has been applied to the realization of recognition and specificity to biomaterials for bioseparations, diagnostic assays, biocatalyses and biosensors. Based on this technology, Liu et al.[10,11] designed and generated a imprinted protein and polymer with glutathione peroxidase(GPx) activity. This bioimprinted enzyme was chemically synthesized with N,S-bis-2,4-dinitrobenzylglutathione(GSH-2DNP) as an imprinting template, and egg albumin as imprinted matrix. In order to generate a better GPx mimic, we changed the imprinted matrix and the template, respectively. We used human serum albumin(HSA) as matrix protein instead of egg albumin because human serum albumin has no antigenicity to human being and it is more applicable in medicine and the use of S-substituted dinitrophenyl glutathione (GSH-S-DNP) instead of GSH-2DNP could increase the affinity of the imprinted protein for the substrate(GSH) because the modification of GSH by some hydrophobic groups could obtain a hydrophobic environment around active site generated by imprinting procedure. Here describes a new bioimprinted enzyme with high GPx activity synthesized via the new imprinting template and imprinted matrix. Moreover, the novel GPx mimic was confirmed to have strong antioxidant ability by a mitochondria damage model.

2 2.1

Materials and Methods Materials

Human serum albumin(HSA), glutathione, glutathione reductase, nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt(NADPH), 5,5′-dithiobis(2-nitrobenzoic acid), 1-chloro-2,4-nitrobenzene(CDNB), ethylenediamine tetraacetic acid(EDTA) were purchased from Sigma. Sephadex G-25 was from Pharmacia.

2.2 Synthesis and Characterization of S-Substituted Dinitrophenyl Glutathione(GSH-S-DNP) GSH(1.02 g, 3.32 mmol/L) in a NaOH(10 mL, 1.0 mol/L) solution was incubated in ice water. 2,4-Dinitrochlorobenzene

Scheme 2

+

GSSG+NADPH+H

GPx mimic

GSSG+2H2O

GSSG reductase

(DNCB, 0.67 g, 3.6 mmol/L) in alcohol was slowly put into the solution of GSH. Then the solution was kept at 0 °C for 10 min and the pH value was adjusted to 4 by dilute HCl. The yellow crystal was produced. Then it was recrystallized from boiling water to give the GSH-S-DNP(0.7 g, yield 76%). Characterization of GSH-S-DNP was performed with infrared spectrometry[12] and elemental analysis.

2.3 Preparation of GSH-imprinted Protein(GSHHSA) Human serum albumin(HSA, 4 mL of a 2 mg/mL solution) was dissolve in 0.5 mol/L NaOH and stirred at room temperature for 1 h. The imprinting molecule, GSH-S-DNP(40 mg), was added to the protein solution and stirred for 3 h. The pH value was then adjusted to 8.0 with 0.1 mol/L HCl and glutaraldehyde solution(1%, 100 μL) was added to it. The reaction mixture was stirred at 4 °C for 16 h and dialyzed for 48 h against 50 mmol/L phosphate buffer, pH=7.0.

2.4 Preparation and GPx Activity of GSH-Imprinted Selenium-Containing Protein(Se-GSH-HSA) The dialyzed solution was purified by Sephadex G-25 gel filtration, then activated by adding 10 mL of phenylmethanesulfonyl flouride(20 mg/mL solution in MeCN) and incubated at 25 °C for 3 h. A portion of 1 mol/L NaHSe(100 mL, 100 mmol/L) prepared according to the method of Xu et al.[13] [Eq.(1)] was added to the activated solution, which incubated at 40 °C for 36 h under nitrogen atmosphere(Scheme 2). 2NaHSe+Na2B4O7+14H2 (1) 4NaBH4+2Se+7H2O The solution was oxidized by air at 4 °C for 16 h and was centrifuged(10000g, 30 min) to remove the solid selenium(Se). The crude imprinted protein was purified by gel filtration on a Sephadex G-25 column, and the first peak was collected, the crosslinked protein under this peak was named for Se-GSH-HSA. Human serum albumin(HSA) was thus converted into selenium-containing protein by the same method. The selenoprotein was purified by Sephadex G-25 gel filtration

Synthetic procedure of selenoproteins

chromatography. The first peak was named for non-printed protein containing selenium(Se-HSA). Se-GSH-HSA concentration was determined by Bradford method[14] with BSA as the standard. The GPx activity was measured according to Wilson’s method[15,16][Eq.(2) and Eq.(3)]. The activity unit is defined as the amount of enzyme that catalyzes 1 μmol of NADPH per minute. The specific activity is expressed in U/μmol of enzyme. 2GSH+H2O2

259

(2) +

2GSH+NADP

(3)

The selenium content was measured as described in references [17,18]. Briefly, the mimic was treated with excess sodium borohydride in phosphate-buffered saline buffer under nitrogen gas for 30 min and then 1 mol/L imidazole was added to the solution to quench the excessive reducing agent. After adusting to pH=7 with 1 mol/L HCl, 100 μL of the solution was taken and added to 5 mL of 0.1 mmol/L 5,5’-dithiobis (2-nitrobenzoic acid). The content of selenol in the mimic was determined from the absorption of 3-carboxy-4-nitrobenzenethiolate at 410 nm(ε=11400 L·mol–1·cm–1).

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2.5 Determination of Binding Se-HSA and Se-GSH-HSA

Constant

of

Binding constant of Se-HSA and Se-GSH-HSA was assessed by quenching the fluorescence of the seleniumcontaining proteins with GSH[19]. Samples were adjusted to an A280 nm(less than 0.05) in a final volume of 4 mL with buffer[20 mmol/L phosphate buffer solution(PBS), pH=7.4]. The measurement was performed with an RF-5301PC spectrofluorophotometer(Shimazu) at λex=295 nm and λem=345 nm. Samples were quenched with GSH with gradually increased concentrations. The binding constant for GSH of the printed protein without selenolation(GSH-HSA) was also determined by the same method.

2.6 Determination of Optimal pH Value and Temperature for Se-GSH-HSA The optimal pH value and optimal temperature of GPx activity of Se-GSH-HSA were measured with the same method as the GPx(H2O2) activity assay. The initial rates were measured at the concentrations of 1 mmol/L GSH and 1 mmol/L H2O2. The pH value of buffer was changed in determining the initial rate of the reaction to obtain the optimal pH condition for Se-GSH-HSA reaction. Similarly, the optimal temperature for Se-GSH-HSA catalyzed reaction was determined at different temperatures.

2.7

Kinetics of Se-GSH-HSA

The assay of kinetics of the mimic was similar to that of native GPx[20]. The initial rates were measured by observing the change of NADPH absorption at 340 nm at several concentrations of one substrate while the concentration of the other substrate was kept constant. All kinetic experiments were performed in 700 μL of the reaction solution containing 50 mmol/L potassium phosphate buffer(pH=7.0), 1 mmol/L EDTA, 1 unit of glutathione reductase, 0.25 mmol/L NADPH, and the appropriate concentrations of GSH, H2O2, and mimic. The mimic was pre-incubated with GSH, NADPH, and glutathione reductase. The reaction was initiated by the addition of appropriate concentrations of H2O2. The noncatalytic reaction affecting the measurement of the initial rate was taken into account and the rate of noncatalytic reaction was subtracted to obtain exact kinetic value. Kinetic data were analyzed by double reciprocal plotting. The relevant steady-state rate equation is shown as follows[Eq.(4)]: kcat[GSH][H2O2] ν0 = GSH (4) H O [E]0 KM [GSH]+KM2 2[H2O2]+[GSH][H2O2] whereν0 is the initial catalytic rate, [E]0 is the initial enzyme mimic concentration, kcat is the pseudo first-order rate conGSH H O stant, KM and KM2 2 are the apparent Michaelis constants of GSH and H2O2, respectively.

2.8 Protection of Mitochondria from Oxidative Damage by Se-GSH-HSA Bovine heart mitochondria were isolated from fresh

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bovine heart according to ref. [21] and suspended in 0.25 mol/L sucrose, 10 mmol/L EDTA and 25 mmol/L HEPES-NaOH buffer(pH=7.4), and maintained at 0 °C. The concentration of the mitochondria proteins was determined by Coomassie brilliant blue[14] with bovine serum albumin as the standard. The incubation mixture consisted of 0.125 mol/L KCl, 1 mmol/L MgCl2, 5 mmol/L glutamate, mitochondria(0.5 mg protein/mL), 1 μmol/L GSH, and appropriate enzyme mimic in 10 mmol/L potassium phosphate buffer(pH=7.4, 37 °C). Thiobarbituric acid(TBA) reactive substances(TBARS) and swelling of mitochondria were determined at some intervals after addition of 0.5 mmol/L ascorbate and 12.5 μmol/L ferrous sulfate. Damage experiments were done without enzyme mimic; control experiments were performed without enzyme mimic, ascorbate and ferrous sulfate. TBARS content in ferrous sulfate/ascorbate-treated mitochondria was analyzed by TBA assay[22]. Swelling of mitochondria was assayed as described by Yu et al.[23]. The swelling of mitochondria was measured as the decrease in turbidity of the reaction mixture at 520 nm. The decrease of the absorbance indicates an increase in the mitochondria swelling and a decrease in the mitochondria integrity. The results were analyzed by one way analysis of variance (ANOVA) followed by Tukey multiple comparison tests to identify individual differences among groups when the ANOVA was significant. All the values were expressed as means±SE (standard error of the mean). P